The goal of this project is to understand the molecular etiology of human cancers, and in particular, to understand the molecular regulation of cell cycle progression and how defects in cell cycle control contribute to the generation of genomic instability and cancer. Intricate molecular regulatory networks control cell cycle checkpoints and these molecular signaling networks respond differently to different types of DNA lesions. Cellular damage results in activation of sensor protein complexes which signal through members of the PI3 protein kinase family that include the ATM (ataxia telangiectasia mutated) and ATR (AT and Rad3 related) kinases. These kinases activate signal transduction cascades that use effector and transducer molecules such as the Chk1 and Chk2 protein kinases and the CDC25 family of protein phosphatases, and thus modulate the activity of the cyclin/cyclin-dependent kinase (Cdk) complexes that regulate transitions through the cell cycle. In addition, these sensor kinase complexes regulate the formation of large regulatory protein complexes (e.g. the Mre11/Rad50/NBS1/BRCA1 complex) which are involved in aspects of DNA damage repair. Precisely which signaling network predominates in a checkpoint response seems to be highly dependent on the nature of the cellular stress and the subsequent lesions generated from that stress, as well as the particular point in the cell cycle when the damage occurs. We have shown that in response to exposure to both ionizing radiation (IR) and oxidative damage from reactive oxygen species (ROS) the early G2 checkpoint is mediated through ATM and results in an inactivation of Cyclin/Cdk complexes. In contrast, we have shown that the cellular response to the inability to untangle daughter chromatids in G2 due to the topoisomerase II poison ICRF193 results in an ATR-mediated G2 decatenation checkpoint. This checkpoint requires the inhibition of Plk1 kinase and results in a sequestering of active Cyclin B1/Cdk1 complexes in the cytoplasm, preventing their normal nuclear functions that drive cells into M phase. We are utilizing large-scale gene expression analysis of cellular responses to DNA damaging agents in an attempt to gain additional insight into cell cycle checkpoint events. In recent studies we are examining the cellular responses to exposures to IR, UV radiation, and ROS in both normal and ATM-deficient diploid human fibroblasts, as well as responses to IR exposures to both normal and ATM-deficient human lympoblasts. We have been able to identify subsets of genes whose expression levels can differentiate between IR, UV radiation and ROS exposures, both ATM-dependent and ATM-independent, and which may serve as marker genes for exposures to these different damaging agents. We are interested in extending these analyses into cells from individuals with the familial genomic instability syndromes Nijmegen Breakage Syndrome (NBS) and Bloom?s Syndrome (BS).